Top Down and Bottom Up Analysis of Proteins (Focusing on Quantitative Aspects)

In recent years two main strategies have been evolved for quantitative proteomics approaches. ‘Top down’ strategies start with a proteome composed of intact proteins. After protein separation to reduce the complexity, the less complex protein fractions will be analysed by mass spectrometric identification techniques following enzymatic cleavage. The quantitation is performed either at the protein level by image analysis or at the peptide level using mass spectrometry, often in combination with stable isotopic labelling techniques like SILAC or ICPL. ‘Bottom up’ strategies, starting with the proteome cleaved into peptides, rely on efficient (multidimensional) peptide separation techniques and identification and quantitation by mass spectrometry. Here too, either label-free or stable isotope labelling approaches (iTRAQ or TMT) are commonly used. However, bottom up proteomics strategies suffer from the loss of the connection between a particular identified peptide and the protein from which it has been derived. This means that important biological features like posttranslational modifications, isoforms and degradation events are difficult to detect.

However, despite these limitations, the increasing demand for monitoring particular interesting proteins quantitatively with high sensitivity has led one particular bottom up proteomics technique, selected reaction monitoring (SRM), to become a most promising tool for targeted analysis in systems biology and biomarker validation.